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Honey is a precious and sustainable natural resource. Ancient literatures belonging to early civilizations mention the use of honey by their physicians to treat patients with different symptoms, injuries or infections. The past few decades have witnessed various laboratories across the globe reporting their findings about the antimicrobial, wound-healing, anti-inflammation, anti-oxidant and anti-tumour effects of natural honey. Unfortunately, its use in modern medicine is severely limited. Scientific community is largely sceptical of its curative impact. The complex chemical composition and different beneficial properties of honey vary with its floral origin. Recently it has come into focus that honey has a very promising role in ‘green nanotechnology’. Nanotechnology offers the opportunity to better exploit the potential of many natural compounds for application in nanomedicine. However, the classic nanotechnology synthetic approaches are often hazardous. Many nations are hence focusing on safer, easier, eco-friendly synthesis methods, using natural resources like honey. But in India, only a handful of laboratories are interested in exploring the immense valuable prospects of different varieties of honey available in different states.Every year only 30-40% of India’s natural honey is harvested for sale and export, while the rest simply dries up. This is a huge waste of a valuable research resource that holds great promise for the pharmaceutical industry and in sustainable health management.

Action observation training (AOT) is a therapeutic approach used in stroke rehabilitation. Videos form the core of AOT, and knowledge of constituent parameters is essential to make the intervention robust and generalizable. Currently, there is a dearth of available information on video parameters to be used for AOT. Our purpose was to identify and describe the parameters that constitute AOT videos for stroke rehabilitation. Electronic databases like PubMed, CINAHL, Scopus, Web of Science, ProQuest, and Ovid SP from inception to date according to PRISMA-ScR guidelines. Title, abstract, and full-text screening were done independently by two authors, with a third author for conflict resolution. Data on video parameters like length, quality, perspective, speed, screen size and distance, sound, and control videos were extracted. Seventy studies were included in this review. The most-reported parameters were video length (85.71%) and perspective of view (62.85%). Movement speed (7.14%) and sound (8.57%) were the least reported. Static landscapes or geometrical patterns were found suitable as control videos. Most video parameters except for length and perspective of view remain underreported in AOT protocols. Future studies with better descriptions of video parameters are required for comprehensive AOT interventions and result generalisation.IMPLICATIONS FOR REHABILITATIONVideos shorter than 5 min may be preferred during action observation training (AOT) intervention in post-stroke.Egocentric view may be better for upper limb dexterity function and allocentric view for gross actions like walking.Choice of video disseminating device depends on its dimension as well as observer distance.Movement speed, video sound, and quality must be considered to obtain more comprehensive AOT videos. Videos shorter than 5 min may be preferred during action observation training (AOT) intervention in post-stroke. Egocentric view may be better for upper limb dexterity function and allocentric view for gross actions like walking. Choice of video disseminating device depends on its dimension as well as observer distance. Movement speed, video sound, and quality must be considered to obtain more comprehensive AOT videos.

Iron(III)-phenolate/carboxylate complexes exhibiting photoredox chemistry and photoactivated reactive oxygen species (ROS) generation at their ligand-to-metal charge-transfer (LMCT) bands have emerged as potential strategic tools for photoactivated chemotherapy. Herein, the synthesis, in-depth characterization, photochemical assays, and remarkable red light-induced photocytotoxicities in adenocarcinomic human immortalized human keratinocytes (HaCaT) and alveolar basal epithelial (A549) cells of iron(III)-phenolate/carboxylate complex of molecular formula, [Fe(L1)(L2)] (1), where L1 is bis(3,5 di-tert-butyl-2-hydroxybenzyl)glycine and L2 is 5-(1,2-dithiolan-3-yl)-N-(1,10-phenanthroline-5-yl)pentanamide, and the gold nanocomposite functionalized with complex 1 (1-AuNPs) are reported. There was a significant red shift in the UV-visible absorption band on functionalization of complex 1 to the gold nanoparticles (λmax: 573 nm, 1; λmax: 660 nm, 1-AuNPs), rendering the nanocomposite an ideal candidate for photochemotherapeutic applications. The notable findings in our present studies are (i) the remarkable cytotoxicity of the nanocomposite (1-AuNPs) to A549 (IC50: 0.006 μM) and HaCaT (IC50: 0.0075 μM) cells in red light (600-720 nm, 30 J/cm2) while almost nontoxic (IC50 > 500 μg/mL, 0.053 μM) in the dark, (ii) the nontoxicity of 1-AuNPs to normal human diploid fibroblasts (WI-38) or human peripheral lung epithelial (HPL1D) cells (IC50 > 500 μg/mL, 0.053 μM) both in the dark and red light signifying the target-specific anticancer activity of the nanocomposite, (iii) localization of 1-AuNPs in mitochondria and partly nucleus, (iv) remarkable red light-induced generation of reactive oxygen species (ROS: 1O2, •OH) in vitro, (v) disruption of the mitochondrial membrane due to enhanced oxidative stress, and (vi) caspase 3/7-dependent apoptosis. A similar cytotoxic profile of complex 1 was another key finding of our studies. Overall, our current investigations show a new red light-absorbing iron(III)-phenolate/carboxylate complex-functionalized gold nanocomposite (1-AuNPs) as the emerging next-generation iron-based photochemotherapeutic agent for targeted cancer treatment modality.

Ineffective cancer treatment is implicated in metastasis, recurrence, resistance to chemotherapy and radiotherapy, and evasion of immune surveillance. All these failures occur due to the persistence of cancer stem cells (CSCs) even after rigorous therapy, thereby rendering them as essential targets for cancer management. Contrary to the quiescent nature of CSCs, a gene profiler array disclosed that phosphatidylinositol-3-kinase (PI3K), which is known to be crucial for cell proliferation, differentiation, and survival, was significantly upregulated in CSCs. Since PI3K is modulated by cyclic adenosine 3',5' monophosphate (cAMP), analyses of cAMP regulation revealed that breast CSCs expressed increased levels of phosphodiesterase 4 (PDE4) in contrast to normal stem cells. In accordance, the effects of rolipram, a PDE4 inhibitor, were evaluated on PI3K regulators and signaling. The efficacy of rolipram was compared with paclitaxel, an anticancer drug that is ineffective in obliterating breast CSCs. Analyses of downstream signaling components revealed a switch between cell survival and death, in response to rolipram, specifically of the CSCs. Rolipram-mediated downregulation of PDE4A levels in breast CSCs led to an increase in cAMP levels and protein kinase A (PKA) expression. Subsequently, PKA-mediated upregulation of phosphatase and tensin homolog antagonized the PI3K/AKT/mTOR pathway and led to cell cycle arrest. Interestingly, direct yet noncanonical activation of mTOR by PKA, circumventing the influence of PI3K and AKT, temporally shifted the fate of CSCs toward apoptosis. Rolipram in combination with paclitaxel indicated synergistic consequences, which effectively obliterated CSCs within a tumor, thereby suggesting combinatorial therapy as a sustainable and effective strategy to abrogate breast CSCs for better patient prognosis.

Leishmania, being an intelligent protozoan parasite, modulates the defensive arsenals of the host to create a favorable niche for their survival. When the intracellular parasite is encountered by the host, multimeric complexes of inflammasomes get assembled and activated, thereby leading to genesis of inflammatory response. In order to subvert host defensive strategies, Leishmania utilizes their cyclic adenosine monophosphate (cAMP) and cAMP-induced response to neutralize macrophage oxidative damage. In this chapter, we summarize our current understanding of the mechanisms of inflammasome activation in macrophages and cAMP homeostasis of the parasite, leading to parasite viability within the macrophages and establishment of infection. Furthermore, we took into account, recent progresses in translating these research areas into therapeutic strategies, aimed at combating macrophage associated diseases.

Purpose The objective of this scoping review was to get an overview of barriers emerging across the globe from the pandemic that are likely to increase the level of pre-existing disability status of neurologically challenged populations. Methods Database searches (PubMed/MEDLINE, CINAHL, ProQuest, Ovid, Scopus, and Web of Science) updated to December 2020 were conducted. Articles that identified challenges or barriers to neuro-rehabilitation, impact on disability status and health care services were included. Full-text articles limited to the English language with no restrictions on study design were included. Data was synthesized based on recurrent themes that were identified. Results Thirty-seven studies were included in this review. Neurological populations considered: stroke, multiple sclerosis, amyotrophic lateral sclerosis, parkinson's disease, autism, developmental disabilities, and those who required neurosurgical care. Barriers were grouped into categories as increased disease risk and complications, delayed or restricted access to neuro-rehabilitation, limited hospital access, telerehabilitation limitations, and shutdown of special centers of aid. Conclusions COVID-19 pandemic has given rise to barriers that affect almost every aspect of healthcare and rehabilitation in neurologically challenged populations prompting an increase in their disability level. This can assist policymakers in designing mitigation strategies to minimize the detrimental effects on this vulnerable population.Implications for rehabilitationPandemic has led to the worsening of existing motor and non-motor symptoms, which need to be monitored, assessed and managed medically, and through rehabilitation in neurologically challenged populations.Notable decline of cognition and physical activity in neurologically challenged populations needs to be assessed and efforts to reverse these outcomes should be attempted.Rehabilitation services, hospital care and centers of aid need to be made more accessible for neurologically challenged populations with COVID-19 precautionary measures.Telemedicine and telerehabilitation need to be upgraded to enhance further face to face like interactions and for tracking of progressive disease.

With the identification of novel cAMP binding effecter molecules in Trypanosoma, role of cAMP in kinetopalstida parasites gained an intriguing break through. Despite earlier demonstrations of role of cAMP in survival of Leishmania during macrophage infection, there is essential need to specifically clarify involvement of cAMP in various cellular processes in the parasite. In this context, we sought to gain a comprehensive understanding of the effect of cAMPanalogs and cAMP-phosphodiesterase (PDE) inhibitors on proliferation of log phase parasites. Administration of both hydrolysable (8-pCPT-cAMP) and non-hydrolysable analogs (Sp-8-pCPT-cAMPS) of cAMP resulted in significant decrease of Leishmania proliferation. Amongst the various PDE inhibitors, etazolate was found to be potently anti-proliferative. BrdU cell proliferation and K/N/F-enumeration microscopic study revealed that both cAMP analogues and selective PDE inhibitors resulted in significant cell cycle arrest at G1 phase with reduced S-phase population. Furthermore, careful examination of the flagellar motility patterns revealed significantly reduced coordinated forward flagellar movement of the promastigotes with a concomitant decrease in cellular ATP levels. Alongside, 8-pCPT-cAMP and PDE inhibitors etazolate and trequinsin showed marked reduction in mitochondrial membrane potential. Treatment of etazolate at subcytotoxic concentration to infected macrophages significantly reduced parasite burden and administration of etazolate to Leishmania-infected BALB/c mice showed reduced liver and spleen parasite burden. Collectively, these results imply involvement of cAMP in various crucial processes paving the avenue for developing potent anti-leishmanial agent.Author SummaryLeishmania donovani is the causative agent of fatal Visceral Leishmaniasis. The current available medications are toxic, expensive and require long term daily administrations. With an aim to develop improved therapeutic, components of cAMP homeostasis, particularly cAMP-phosphodiesteares, has been targeted for Leishmania and other kinetoplastid pathogens. cAMP plays diverse roles in functional processes involved in cell division, transition into different stages of the life cycle of Leishmania and motility. In this study, the authors found administration of both hydrolysable and non-hydrolysable analogs of cAMP and certain PDE inhibitors resulted in remarkable decrease proliferation with considerable cytopathic impact on promastigotes. The mammalian phosphodiestearse inhibitor etazolate caused significant reduction in parasite load in L. donovani infected macrophages and demonstrated considerable reduction of liver and spleen parasite burden in in vivo mouse infection model. The study suggested that etazolate, with its slightest impact on mammalian host, can be repurposed for developing effective anti-leishmanials.

Eight indolo[3,2-a]phenanthridine derivatives have been synthesized in a regioselective manner involving intramolecular Heck-type arylation as a key step. The compounds display interesting photophysical proprties and hence evaluated for their ability to interact with ct-DNA. Preliminary biophysical studies via UV and Fluorescence spectrophotometric titration with ct-DNA, and dye displacement studies with well known intercalator ethidium bromide and the groove binder Hoechst 33,258 reveal that the binding mode is probably minor groove binding. The prepared indolophenanthridine derivatives have also been evaluated as anti-leishmanial agents for the first time. MTT-assays for cell cytotoxicity against Leishmania promastigotes and Leishmania amastigotes were studied with the compounds 10b-f, 12-14 for the determination of their IC

Exposure of Leishmania donovani to macrophage phagolysosome conditions (PC) (37°C and pH 5.5) led to increased intracellular cAMP and cAMP-mediated responses, which help in intra-macrophage survival pre-requisite for infectivity. In the absence of typical orthologs for G-proteins and G-protein coupled receptors, we sought to study the precise mechanisms for positive modulation of cAMP production during exposure to PC. Amongst two promastigote-stage specific membrane bound receptor adenylate cyclases (LdRAC-A and LdRAC-B), LdRAC-A appeared to function as a major cAMP generator following PC exposure. Pyrophosphate (PPi), an energy storage compound as well as a by-product of cAMP biosynthesis by adenylate cyclise, was found to be decreased following PC exposure. This may be due to microtubule and microfilament-driven translocation of acidocalcisomes near plasma membrane vicinity with concomitant increase of acidocalcisome membrane pyrophosphatase (LdV-H+PPase) and acidocalcisomal soluble pyrophosphatase (LdVSP1). Episomal over-expression and conditional silencing demonstrated regulatory role of V-H+PPase on cAMP trigger and consequent induction of resistance to macrophage-derived pro-oxidants and parasite killing. Furthermore, immunofluorescence analysis revealed possible co-localization of LdV-H+PPase and LdRAC-A during PC exposure. Collectively, these results suggest that translocation of acidocalcisome in membrane vicinity functions as a trigger for LdRAC-A-driven cAMP generation through depletion of PPi pool by LdV-H+PPase.

Pregnane X receptor (PXR) is a major transcriptional regulator of xenobiotic metabolism and transport pathways in the liver and intestines, which are critical for protecting organisms against potentially harmful xenobiotic and endobiotic compounds. Inadvertent activation of drug metabolism pathways through PXR is known to contribute to drug resistance, adverse drug-drug interactions, and drug toxicity in humans. In both humans and rodents, PXR has been implicated in non-alcoholic fatty liver disease, diabetes, obesity, inflammatory bowel disease, and cancer. Because of PXR's important functions, it has been a therapeutic target of interest for a long time. More recent mechanistic studies have shown that PXR is modulated by multiple PTMs. Herein we provide the first investigation of the role of acetylation in modulating PXR activity. Through LC-MS/MS analysis, we identified lysine 109 (K109) in the hinge as PXR's major acetylation site. Using various biochemical and cell-based assays, we show that PXR's acetylation status and transcriptional activity are modulated by E1A binding protein (p300) and sirtuin 1 (SIRT1). Based on analysis of acetylation site mutants, we found that acetylation at K109 represses PXR transcriptional activity. The mechanism involves loss of RXRα dimerization and reduced binding to cognate DNA response elements. This mechanism may represent a promising therapeutic target using modulators of PXR acetylation levels. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

In the last few years, nanoparticles and nanocomposites have emerged as one of the promising candidates to scientists from various fields because of their immense potential to revolutionize science and technology. The nanoscale particles and composites are synthesized with a broad range of metals like gold, silver, iron, metal oxides and semiconductors. They are effective for water filtration, as a therapeutic agent, a very important agent for targeted drug delivery and also of immense importance in biomedical applications like Magnetic Resonance Imaging (MRI). The nanoscale particles have a wide range and have the potential to be used for the betterment of biomedical research, human health, and environment. Though these nanoscale materials are synthesized widely all over the world with various metals, carbon and graphene and other elements for research purposes and to understand their applications, the biological issues of toxicity associated with these materials and its impact on human health and environment are grossly unexplored. Detailed understanding of the factors regulating toxicity is lacking. A complete toxicological profile of these nanocomposites will ensure effective translation for a market available drug through clinical trial and other nano-based products. This chapter deals with the synthesis of nanocomposites, their applications and toxicological evaluation of the same in terms of human application.

Intracellular cAMP level and cAMP mediated responses are elevated when Leishmania are exposed to macrophage phagolysosome conditions (37 °C and pH 5.5). Phosphodiesterases play major role in cAMP regulation and in the present study we have cloned and characterized a 2.1 kb cytosolic isoform of phosphodiesterase from Leishmania donovani (LdPDED) which plays important role in cAMP homeostasis when the promastigotes are exposed to macrophage phagolysome conditions for converting to axenic amastigotes. Domain characterization suggested the presence of two pseudo-substrate sites similar to the ones present in the regulatory subunit of cAMP-dependent protein kinase A (PKA) and a putative PKA phosphorylation site at T 708 of C-terminus of LdPDED. Deletion constructs and site directed mutagenesis revealed the ability of LdPDED to interact with L. donovani PKA catalytic subunits (LdPKAC1 and LdPKAC2) resulting in inhibition of kinase activity in one hand and increase of phosphodiesterase activity through PKA mediated phosphorylation at putative phosphorylation site on the other hand. This study therefore identifies a unique phosphodiesterase in L. donovani which appears to regulate cAMP-dependent PKA signaling through a two way process.

The functional role of the C2 insert of nonmuscle myosin II-C (NM II-C) is poorly understood. Here, we report for the first time that the expression of the C2 insert-containing isoform, NM II-C1C2, is inducible in Neuro-2a cells during differentiation both at mRNA and protein levels. Immunoblot and RT-PCR analysis reveal that expression of NM II-C1C2 peaks between days 3 and 6 of differentiation. Localization of NM II-C1C2 in Neuro-2a cells suggests that the C2 insert-containing isoform is localized in the cytosol and along the neurites, specifically at the adherence point to substratum. Inhibition of endogenous NM II-C1C2 using siRNA decreases the neurite length by 43% compared with control cells treated with nonspecific siRNA. Time lapse image analysis reveals that neurites of C2-siRNA-treated cells have a net negative change in neurite length per minute, leading to a reduction of overall neurite length. During neuritogenesis, NM II-C1C2 can interact and colocalize with β1-integrin in neurites. Altogether, these studies indicate that NM II-C1C2 may be involved in stabilizing neurites by maintaining their structure at adhesion sites.

cAMP-mediated responses act as modulators of environmental sensing and cellular differentiation of many kinetoplastidae parasites including Leishmania. Although cAMP synthesizing (adenylate cyclase) and degrading (phosphodiesterase) enzymes have been cloned and characterized from Leishmania, no cAMP-binding effector molecule has yet been identified from this parasite. In this study, a regulatory subunit of cAMP-dependent protein kinase (Ldpkar1), homologous to mammalian class I cAMP-dependent protein kinase regulatory subunit, has been identified from L. donovani. Further characterization suggested possible interaction of LdPKAR1 with PKA catalytic subunits and inhibition of PKA activity. This PKA regulatory subunit is expressed in all life cycle stages and its expression attained maximum level in stationary phase promastigotes, which are biochemically similar to the infective metacyclic promastigotes. Starvation condition, the trigger for metacyclogenesis in the parasite, elevates LdPKAR1 expression and under starvation condition promastigotes overexpressing Ldpkar1 attained metacyclic features earlier than normal cells. Furthermore, Ldpkar1 overexpression accelerates autophagy, a starvation-induced cytological event necessary for metacyclogenesis and amastigote formation. Conditional silencing of Ldpkar1 delays the induction of autophagy in the parasite. The study, for the first time, reports the identification of a functional cAMP-binding effector molecule from Leishmania that may modulate important cytological events affecting metacyclogenesis.

The nuclear receptor pregnane X receptor (PXR) is activated by a range of xenochemicals, including chemotherapeutic drugs, and has been suggested to play a role in the development of tumor cell resistance to anticancer drugs. PXR also has been implicated as a regulator of the growth and apoptosis of colon tumors. Here, we have used a xenograft model of colon cancer to define a molecular mechanism that might underlie PXR-driven colon tumor growth and malignancy. Activation of PXR was found to be sufficient to enhance the neoplastic characteristics, including cell growth, invasion, and metastasis, of both human colon tumor cell lines and primary human colon cancer tissue xenografted into immunodeficient mice. Furthermore, we were able to show that this PXR-mediated phenotype required FGF19 signaling. PXR bound to the FGF19 promoter in both human colon tumor cells and “normal” intestinal crypt cells. However, while both cell types proliferated in response to PXR ligands, the FGF19 promoter was activated by PXR only in cancer cells. Taken together, these data indicate that colon cancer growth in the presence of a specific PXR ligand results from tumor-specific induction of FGF19. These observations may lead to improved therapeutic regimens for colon carcinomas.

Pregnane x receptor (PXR, NR1I2) was originally characterized as a broad spectrum entero-hepatic xenobiotic ‘sensor’ and master-regulator of drug inducible gene expression. A compelling description of ligand-mediated gene activation has been unveiled in the last decade that firmly establishes this receptor’s central role in the metabolism and transport of xenobiotics in mammals. Interestingly, pharmacotherapy with potent PXR ligands produces several profound side effects including decreased capacities for gluconeogenesis, lipid metabolism, and inflammation; likely due to PXR-mediated repression of gene expression programs underlying these pivotal physiological functions. An integrated model is emerging that reveals a sophisticated interplay between ligand binding and the ubiquitylation, phosphorylation, SUMOylation, and acetylation status of this important nuclear receptor protein. These discoveries point to a key role for the post-translational modification of PXR in the selective suppression of gene expression, and open the door to the study of completely new modes of regulation of the biological activity of PXR.

Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

Eukaryotic initiation factor 6 (eIF6), a highly conserved protein from yeast to mammals, is essential for 60 S ribosome biogenesis and assembly. Both yeast and mammalian eIF6 are phosphorylated at Ser-174 and Ser-175 by the nuclear isoform of casein kinase 1 (CK1). The molecular basis of eIF6 phosphorylation, however, remains elusive. In the present work, we show that subcellular distribution of eIF6 in the nuclei and the cytoplasm of mammalian cells is mediated by dephosphorylation and phosphorylation, respectively. This nucleo-cytoplasmic shuttling is dependent on the phosphorylation status at Ser-174 and Ser-175 of eIF6. We demonstrate that Ca(2+)-activated calcineurin phosphatase binds to and promotes nuclear localization of eIF6. Increase in intracellular concentration of Ca(2+) leads to rapid translocation of eIF6 from the cytoplasm to the nucleus, an event that is blocked by specific calcineurin inhibitors cyclosporin A or FK520. Nuclear export of eIF6 is regulated by phosphorylation at Ser-174 and Ser-175 by the nuclear isoform of CK1. Mutation of eIF6 at the phosphorylatable Ser-174 and Ser-175 to alanine or treatment of cells with the CK1 inhibitor, D4476 inhibits nuclear export of eIF6 and results in nuclear accumulation of eIF6. Together, these results establish eIF6 as a substrate for calcineurin and suggest a novel paradigm for calcineurin function in 60 S ribosome biogenesis via regulating the nuclear accumulation of eIF6.

Even though the human parasite Leishmania donovani encounters tremendous oxidative burst during macrophage invasion, a set of parasites survives and proliferates intracellularly, leading to transformation from promastigote to amastigote form and disease manifestation. The striking shifts in temperature (from 22 degrees C in the insect gut to 37 degrees C in the mammalian host) and pH (7.2 in the insect gut to 5.5 in the parasitophorous vacuole of macrophages) are the key environmental triggers for differentiation as these cause an arrest in the G1 stage of the cell cycle and initiate transformation. Using an established in vitro culture and differentiation system our study demonstrates that the differentiation-triggering environment induces resistance to oxidative damage and consequently enhances infectivity. Differentiation conditions caused a three- to fourfold elevation in cAMP level as well as cAMP-dependent protein kinase activity. Similar to stress exposure, positive modulation of intracellular cAMP resulted in blockage of cell cycle progression and induction of resistance against oxidative damage. Resistance against pro-oxidants from either stress or cAMP may be associated with upregulation of the expression of three major antioxidant genes, peroxidoxin 1, trypanothione reductase, and superoxide dismutase A. Positive modulation of the intracellular cAMP response enables cells to resist the cytotoxic effects of pro-oxidants. In contrast, downregulation of intracellular cAMP by overexpression of cAMP phosphodiesterase A resulted in a decrease in resistance against oxidative damage and reduced infectivity toward activated macrophages. This study for the first time reveals the importance of cAMP response in the life cycle and infectivity of the Leishmania parasite.

Persistence of intracellular infection depends on the exploitation of factors that negatively regulate the host immune response. In this study, we elucidated the role of macrophage PGE2, an immunoregulatory lipid, in successful survival of Leishmania donovani, causative agent of the fatal visceral leishmaniasis. PGE2 production was induced during infection and resulted in increased cAMP level in peritoneal macrophages through G protein–coupled E-series prostanoid (EP) receptors. Among four different EPs (EP1–4), infection upregulated the expression of only EP2, and individual administration of either EP2-specific agonist, butaprost, or 8-Br–cAMP, a cell-permeable cAMP analog, promoted parasite survival. Inhibition of cAMP also induced generation of reactive oxygen species, an antileishmanial effector molecule. Negative modulation of PGE2 signaling reduced infection-induced anti-inflammatory cytokine polarization and enhanced inflammatory chemokines, CCL3 and CCL5. Effect of PGE2 on cytokine and chemokine production was found to be differentially modulated by cAMP-dependent protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC). PGE2-induced decreases in TNF-α and CCL5 were mediated specifically by PKA, whereas administration of brefeldin A, an EPAC inhibitor, could reverse decreased production of CCL3. Apart from modulating inflammatory/anti-inflammatory balance, PGE2 inhibited antileishmanial IL-17 cytokine production in splenocyte culture. Augmented PGE2 production was also found in splenocytes of infected mice, and administration of EP2 antagonist in mice resulted in reduced liver and spleen parasite burden along with host-favorable T cell response. These results suggest that Leishmania facilitates an immunosuppressive environment in macrophages by PGE2-driven, EP2-mediated cAMP signaling that is differentially regulated by PKA and EPAC.